Monika Kern fl Microbial Diversity course MB!,, Woods Hole 1991 Isolation of Heliobacteriaceae The Heliobacteriaceae are anoxygenic photothrophic bacteria. In 1983 Howard Gest first isolated Heliobacterium chiorum from surface soil and until now only two species Heliobacterium and Heliobacillus are known. The Heliobacteriaceae are distinguished from all other anoxygenic phototrophs because off several aspects: - they contain bacterichlorophyll g with an absorption maximum at 790nm, which differs from various bacteriochlorophylls from green and purple bacteria (vinyl group on ring I of tertrapyrrole like in plant chl a; pyrrol ring II is reduced as in bcl a and b; as esterfying alcohol bchl g contains farnesol which also occurs in the bchl of green sulfur bacteria; hel g shares structural relationship of chl a and bchl), — Heliobacteria seem to be primary soil bacteria, — their green colour is due to the presence of bchl g and the green carotenoid neurosporene, — heliobateria lack differentiated structures like chlorosomes or intracytoplasmic membranes, the photopigment systems are located in the cytoplasmic membrane, — heliobacteria are the only anoxigenic phototrophic bacteria which can font endospores resitent to high temperature and dryness. This year during the Amherst Symposium on Photosynthesis the existence of the endospores could first be demonstrated. Photoheterotrophic growth only occurs under strict anaerobic conditions. They are able to fix molecular nitrogen. Until now heliobacteria have only been isolated from soil especially from paddy soil (Thailand, Tanzania) and they were found to withstand dryness much better than other phototrophic bacteria. They have not yet been isolated from aquatic habitats. Unlike purple nonsulfur bacteria only few carbonsources are photometabolized. Pyruvate is known to be the best substrate, besides pyruvate lactate, acetate (+ bicarbonate), butyrate and malate can be used. Methods For selective enrichment of Heliobacteria dry surface soils (Swope ground, garden soil, forest soil) as well as freshwater from a pond near the bikepath to Falmouth were used. Because Heliobacteria can form heat resistance endospores all samples were pasteurized (15 mm, 80°C) before inoculation of enrichment cultures. All experiments were performed under anaerobic conditions (in the anaerobic hood). Cultures were incubated at 35°C and at about 8000 lux light intensity. Because high light intensities and high C temperatures tend to discourage growth of purple and green bacteria enrichment cultures were also inoculated with not heat treated samples. Media For enrichment cultures media according to Madigan M.T. The Heliobacteriacea, In: The Prokaryotes 2nd edition, in press were used (see appendix). Pyruvate mineral salt medium - source, 4C1 PMS: pyruvate (2g/l) as carbon and electron NH (lg/l) as nitrogen source, — PMS—N: without ammonia, N2 as nitrogen source — without pyruvate, contains acetate (2.7g/l) plus MIS: 4C1 (lg/l), NaHCO3 (lg/l) and NH — AMS—N: MIS without ammonia, N2 as nitrogen source Mixed acid mineral salts media contains a mixture of organic acids as carbon and MAMelectron sources: sodium—acetate lg/ 1 sodium—malate 2g/l sodium—lactate lg/ 1 + 4C1 MAN: NH (lg/l) MAN—N: without ammonia, N2 as nitrogen source Concentrated stock solutions of the following components were sterilized separately and added to the basal medium after autoclaving: phosphates were autoclaved separately, carbon sources, 3 cystein were filtersterilized. vitamines, NaHC0 and After autoclaving all media were cooled under nitrogen and 1mM cystein was added as a reducing agent. Enrichment cultures for the isolation of Heliobacteracea sample media garden soil pasteurized P145, MIS, MAN plus NH4+ or N2 garden soil (no heat) P148-N, P345-N, MAN-N Swope ground pasteurized P145, MIS, MAN plus N1i4+ or N2 Swope ground (no heat) P145-N, MIS—N, MAN—N forest soil pasteurized PMS, ANS, MAN plus or N2 forest soil (no heat) PMS—N, AXS—N, MAN—N freshwater and fresh water 4C1PMS, ANS MAN plus slurry pasteurized NH or N2 Identification of heliobacterial growth For detecting heliobacterial growth spectra were run (range 350 - 1000 nm). Bchl g in vivo shows an absorption maximum around 790 rim. flchl g is very sensitive towards damage by oxygen. To avoid exposure to 02 samples for spectral measurements were within the anaerobic chamber mixed with sucrose (final 60%) and as a reducing agent 0.05% ascorbate was added. Results No growth of Heliobacteria in any of the enrichment cultures could be observed (measurements of spectra, microscopy). In some of the enrichment cultures high gas production occured, probably due to the growth of Clostridia. H2S production in some enrichments inoculated with pateurized samples was probably due to growth of endospore forming sulfate reducers (Desulfotomaculum). After one week two enrichments which were inoculated with garden soil (not pasteurized, media: MIS-N, MAX—N) showed growth of purple non sulfur bacteria accompained by growth of algae. After two and a half week and several transfers to fresh media it seemed that the purple non sulfur bacteria had outcompeted the algae. It turned out that the most abundant purple non sulfur bacteria in this enrichments was Rhodomicrobium vannielii. Rhodomicrobium vannielii multiplies by budding and cells can easily identified under the light microscop. It is the only purple non sulfur bacterium which is able to form exospore like cysts and this may explain its survival in dry soils. References — Gest et al. (1985) Ferns Microbiol Ecol. 31, 317—322 — Madigan, M. T. In: The Prokaryotes, 2nd edition (in press) — Brockmann, N.; Lipinski A. (1983) Arch. Microbiol. —12— 2 as sole nitrogen source usually develop more slowly cultures employing.N than enrichments containing ammonia,, but the use of N2 generally eliminates interfernce from fermentative anaerobes which occasionally overgrow heliobacteria in pyruvate—based enrichments. - Medium I and 2 have been successfully employed for enrichment cul€ure of heliobacteria: - MEDIUM 1 - PYRUVATh MINERAL SALTS (pus) Distilled water — 1 liter Ethylenediaminetetraacetate—Na—salt — 10mg - 1 MgS04.7H20— 200mg Pa-ic CaCl2.2HO — 75mg LNM4C1 — ig 1C2HP04 — 0.9g — - - - Sodium pyruvate — 2.2g - - - - - - - - Trace element solution — 1 ml - - Yeast extract — O.lg Vitamin 12 — 20 pg Adjust pH to 6.B with NaOI{ or 14C1 and sterilize by autoclaving 20 mm. After cooling briefly add to final concentration of 1mM either methionine, cysteine, or thiosulfate and transfer to anaerobic chamber until used. MODIFICATIONS OF MEDIUM PMS 12-IS = flIS minus pyruvate plus 2OmN sodium lactate 4C1. Beadspace of enrichment should be PHS—N PMS minus NIi 2:C0 N (95:5) 3] J}-L MIS PMS minus pyruvate plus 20mM sodium acetate and 0.1% NaHCO MEDIUM 2 - MiXED ACID MINERAL SALTS (HAMS) This enrichment medium avoids the use of pyruvate and employs a variety —13— of organic acids sa potential photoheterotrophic substrates. Distilled water — I liter jtthylenediaminetetraacetate—Na—salt—lOmg • NaCl — 0.4g ‘HgSO4.71120 — 200mg • taCl2.2110 — 75mg • ¶jH4Cl — 0.8 — O.45g — O.3g • Lj(112P04 • fNaRCO3—lg /Trace elements I ml iYeast extract — 0.2g E17—ZO - Vitamin ig Sodium acetate — lg - - - • Sodium malate — 2g Sodium lactate — Ig Adjust p11 to 6.8 and sterilize by autoclaving 20 mm. When partially 3 from a filter sterilized stock solution and add to cooled, add NaIICO final concentration of luft1 either methionine, cysteine, or thiosulfate and transfer to anaerobic chamber until used. MODIFICATIONS OF HANS = 4C1. Neadspace of enrichment should HANS—N HANS minus NN N2:C0 (95:5) be MEDIUM 3 — PYRUVATE—TEAST EXTRACt (pm) ?iEDIUN This medium is suitable for growth of pure cultures of hflgba teria but is unsuitable for enrichment Distilled water — 1 liter — ig —14— HgSO4.7H20 — 200mg CaC12.2H0 —20mg - Na2S03.5H0— 100mg -. - Sodium pyruvate — 2.2g - Yeast extract — 8g - Adjust to p1- 7, autoclave and store in anaerobic chamber until used. TRACE ELEMENTS - -. Distilled water — 1 liter, - ‘- Ethylenediaminetetraacetate—Na—salt—5.2g 2.4110 — ‘-7eCl 1.5g r ZnC12 — 70mg - 4fnCl2.4110 — 100mg - }13B0 — 6mg - 2.6H0— 190mg - -. V CoC1 -- : 1,/CuCl2.2H0 — 17mg -. - - - VNicl2.6uo — 25rng - - - - - — 188mg 3 - - 0CLL—’ V0 VoSO4.2H20 30mg /t5a 2C1Th& LNa2WO4.2110 — 2mg Add compounds in the above order; make sure that the EDTA is fully dissolved before adding remaining components. Store at 4°C. Growth Vessels Growth of pure cultures of heliobacteria can be accomplished in rubber stoppered culture tubes (Bellco anaerobic culture tubes, 18 x 142mm) or in scrth—capped tubes or bottles. Sterile empty vessels can be filled aseptically with sterile prereduced media within the anaerobic chamber and then sealed with stoppers or screw taps before removal. Screw cap tubes or nioni252 0.200 .---1 --.——-I -- No. Wavelength (nm.) Abs. 965.00 0.026 2 857 .00 0.119 3 802.00 0.085 4 591.00 0.059 5 512.00 0.100 6 479.50 0.114 6 A K b 0.100 3 S 4. - 1. 0.000— . I • I • I • I 350.0 675.0 1000 .0 Wavelength (nm.) Created: 10: 32 07/27/91 Data: Original I IOp’n Scan Speed: Fast Slit Width: 1.0 enrichment25 PhadomicyosQean, tann,g 0/ C 0 0 Monika Kern, Juergen Breitung C) Microbial Diversity MBL, Woods Hole 1991 )naerobic growth of bioluminescent bacteria with trimethylantine N—oxide as a terminal electron acceptor: TMAO respiration TMAO: Trimethylamine N-oxide TMA Trimethylamine TMAO and TMA are waste products of protein metabolism. These compounds are very abundant in marine environments. THA for example mainly contributes to the odor of spoiling fish and TMA—assays in general are used to evaluate fish freshness. TMAO is used as a osmolyte by some marine organisms and several marine fish excrete TMAO to eliminate excess nitrogen. In their natural habitats marine bioluminescent bacteria especially those which grow in fish tissues often have to face high TMAO concentrations. For example TMAO concentration in fish tissue is frequently more than 1% of the wet weight. It has been observed that TMAO facilitates anaerobic growth of several bacteria.